Biliary tract cancers are heterogeneous and aggressive tumors usually classified into cholangiocarcinoma (both intrahepatic and extrahepatic) and gallbladder cancer, which display different phenotypic characteristics. Early phases are frequently asymptomatic, so many patients are diagnosed at advanced stages, when therapies are less effectives. Gemcitabine plus cisplatin is the reference regimen for systemic chemotherapy, although other drugs are commonly used (i.e. doxorubicin, sorafenib and regorafenib). However, their success is rather low because of multidrug resistance (MDR) development. Different mechanisms can contribute to the occurrence of chemoresistance, although the over-expression of ATP-binding cassette (ABC) transporters, often mediated by STAT3 and Nrf2 pathways, seems to be mainly involved. These proteins actively pump several lipophilic molecules across cell biomembrane, thereby increasing drug efflux from cells. Therefore, using ABC-transporter inhibitors (namely chemosensitizers) in association with cytotoxic drugs represents an emerging approach to overcome MDR. In this context, in the present study the ability of two natural sesquiterpenes, ß-caryophyllene (CRY) and ß-caryophyllene oxide (CRYO), to act as chemosensitizers in cholangiocarcinoma and gallbladder cancer cells will be evaluated. Cytotoxicity of the test substances, alone or in combination with anticancer drugs, will be evaluated by MTT assay. Also, selected cell lines will be characterized for the ABC transporter, STAT3 and Nrf2 expression. The possible interference of CRY and CRYO with expression and function of ABC-transporters and STAT3/Nrf2 pathway will be studied by western blot analysis, as a potential chemosensitizing mechanism. The expected results will enable to characterize the possible usefulness of chemosensitizing caryophyllane sesquiterpenes in cholangiocarcinoma and gallbladder cells, thus suggesting a new strategy to support chemotherapy and overcome chemoresistance.
Present research aims to collect a series of results applicable in the battle against biliary tract cancers, a very aggressive malignancy progressively increasing in incidence and death rate. These types of cancer are often diagnosed at later stages when surgery is no longer possible, and chemotherapy remains the only possible therapy [1]. Unfortunately, the response of patients to this treatment is very low due to the occurrence of multidrug resistance. Among the different mechanisms of chemoresistance, the ABC transporter overexpression seems to be the most common [2]. Different isoforms of these pumps can be expressed in biliary tract tumours and their distribution could differ depending on the tumour location (i.e. intra and extrahepatic) [3]. However, current knowledge is scarce and sometimes confused. Most of the drugs used for the treatment of biliary tract cancers are effluxed by ABC transporters. Particularly, MDR1 and MRPs transporters seem to be mainly involved in the efflux of both standard (gemcitabine, cisplatin, doxorubicin) and targeted (sorafenib) therapies [4]. Therefore, it is essential to understand the molecular basis of the strong chemoresistance of biliary tract cancers, considering also that after failure of first-line therapies, there is no further chemotherapy that will improve the survival [5]. In this context, the identification of the main ABC pumps involved in the different type of biliary tract cancers will increase our scientific knowledge so allowing us to recognize their distinctive nature. Moreover, the identification of the characteristic ABC pumps for each kind of tumour could be useful as a potential biomarker that might be used to predict the therapeutic efficacy of drugs in those diseases.
Furthermore, the present project aims to the identification of new chemosensitizing agents able to sensitize CCA and gallbladder carcinoma cancer cells to anticancer treatment by modulating ABC transporter function and expression. Indeed, although drugs already approved for human use (e.g. verapamil, diclofenac) have showed chemosensitizing properties in combination with anticancer drugs, none of them has been successful in clinical trials yet; moreover, most of them determine serious adverse events (e.g. cardiotoxicity) at the doses effective to reverse multidrug resistance [6, 7]. Achieving this goal could allow to design a new combination treatment for human biliary tract cancer, based on the co-administration of antitumor drugs and natural sesquiterpenes. The combination therapy represents a promising approach for restoring drug sensitivity. Blocking drug extrusion from cancer cell could restore its accumulation and the chemotherapy efficacy. Furthermore, due to the combination with the chemosensitizer, lower doses of anticancer drug are required, so the adverse effects of chemotherapy can be reduced. Noteworthy, both CRY and CRYO are considered safe by Authority Agency when used as food additive, respectively [8]. Moreover, previous in vivo experiments showed a protective role of CRY toward doxorubicin cardiotoxicity [9].
Among the advantages of a combination therapy, besides the reduction of anticancer drug toxicity, the simultaneous blockage of distinct key molecules in oncogenic signalling network appears one of the most promising approaches to conquer cancer. In this respect, present research will investigate the potential modulation by both CRY and CRYO of STAT3/Nrf2 pathway, implicated in ABC transporter overexpression and in cancer cell proliferation [10, 11]. The potential inhibition of that signalling could be also useful to avoid the induction of epithelial-mesenchymal transition (EMT) phenotype, which is associated with cancer malignancy [12]. Another advantage of the combination therapy could be the prevention of drug resistance acquisition. Indeed, although chemoresistance is sometimes present before the treatment, it mostly occurs after the pharmacological therapy. The prophylactic use of the combination treatment could reduce this occurrence.
Taken together, the results acquired within present research project could provide a new approach to overcome MDR phenomenon and consequently to improve the quality of life of patients by increasing the survival expectations.
References
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